Regulation of T cell-mediated immunity by tryptophan

ABSTRACT

A mechanism of macrophage-induced T cell suppression is the selective elimination of tryptophan and/or increase in one or more tryptophan metabolites within the local macrophage microenvironment. Studies demonstrate that expression of IDO can serve as a marker of suppression of T cell activation, and may play a significant role in allogeneic pregnancy and therefore other types of transplantation, and that inhibitors of IDO can be used to activate T cells and therefore enhance T cell activation when the T cells are suppressed by pregnancy, malignancy or a virus such as HIV. Inhibiting tryptophan degradation (and thereby increasing tryptophan concentration while decreasing tryptophan metabolite concentration), or supplementing tryptophan concentration, can therefore be used in addition to, or in place of, inhibitors of IDO. Similarly, increasing tryptophan degradation (thereby, decreasing tryptophan concentration and increasing tryptophan metabolite concentration), for example, by increasing IDO concentration or IDO activity, can suppress T cells. Although described particularly with reference to IDO regulation, one can instead manipulate local tryptophan concentrations, and/or modulate the activity of the high affinity tryptophan transporter, and/or administer other tryptophan degrading enzymes. Regulation can be further manipulated using cytokines such as macrophage colony stimulating factor, interferon gamma, alone or in combination with antigen or other cytokines.

This application is a divisional application of U.S. application Ser.No. 10/112,362, filed on Mar. 28, 2002, entitled “Regulation of TCell-Mediated Immunity by Tryptophan” (now U.S. Pat. No. 7,160,539),which is a divisional of U.S. application Ser. No. 09/206,274, filed onDec. 4, 1998, entitled “Regulation of T Cell-Mediated Immunity byTryptophan (now U.S. Pat. No. 6,451,840, issued Sep. 17, 2002; whichclaims priority to U.S. Ser. No. 60/067,610 entitled “Regulation of TCell Activation” filed Dec. 5, 1997; U.S. Ser. No. 60/080,384 entitled“Regulation of Pregnancy” filed Apr. 1, 1998; and U.S. Ser. No.60/080,380 entitled “IDO Inhibitors for Use as Antiviral Agents” filedApr. 1, 1998, by David Munn and Andrew Mellor. U.S. application Ser. No.09/727,055, filed Nov. 30, 2000, entitled “Regulation of T Cell-MediatedImmunity by Tryptophan” (now U.S. Pat. No. 6,482,416, issued Nov. 19,2002) is a divisional of U.S. application Ser. No. 09/206,274 filed onDec. 4, 1998, entitled “Regulation of T Cell-Mediated Immunity byTryptophan (now U.S. Pat. No. 6,451,840, issued Sep. 17, 2002). All ofthese applications are incorporated by reference.

This invention was made in part with government support under Grant No.HD037800 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in this invention.

The present invention is generally in the area of regulation of T cellactivation using modulators of the enzyme indoleamine 2,3-dioxygenase(IDO) which is used by immunosuppressive antigen-presenting cells suchas tissue macrophages and placental trophoblasts to prevent T cells fromactivating in response to antigens presented by these cells. Modulationof the enzyme activity can therefore be used to affect pregnancy,infection by certain viruses such as HIV, and inflammation. Morespecifically, the present invention includes altering maternal toleranceof pregnancy using modulators of the enzyme indoleamine 2,3-dioxygenase(IDO) which is used by immunosuppressive antigen-presenting cells suchas tissue macrophages and placental trophoblasts to prevent T cells fromactivating in response to antigens presented by these cells.

BACKGROUND OF THE INVENTION

Traditionally, the “professional” antigen-presenting cells (APCs) of themyeloid lineage, dendritic cells and macrophages, have been viewedprimarily as accessory cells, functioning simply to assist T cellactivation. Recently, however, it has become clear that myeloid-lineageAPCs exert a profound influence on T cells, regulating both the natureof the response (humoral versus cellular immunity) and, in some cases,even whether a response occurs at all (activation versus anergy). Thishas been the subject of recent reviews by Fearon and Locksley (Science1996; 272:50-54) and Trinchieri and Gerosa (J. Leukocyte Biol. 1996;59:505-511). Currently, the biology of myeloid-lineage APCs is not wellunderstood. Dendritic cells and macrophages appear to derive from acommon progenitor in the myelomonocytic lineage (Szabolcs, et al. Blood1996; 87:4520-4530), but their markedly different functionalcharacteristics are determined during a complex process of hematopoieticdifferentiation, which continues well after their exit from the bonemarrow (Thomas, et al. Stem Cells 1996; 14:196-206). Hematopoieticdifferentiation has traditionally fallen outside the purview ofclassical immunology.

Macrophages enter the tissues at the immature stage of circulatingmonocytes. Using in vitro models, it has been shown that the cytokinemilieu which they encounter at this early stage determines the phenotypewhich they will subsequently adopt. Under the influence of certaincytokines (in humans, usually GM-CFS plus IL-4 or TNF), monocytesdifferentiate in vitro into cells which closely resemble dendritic cells(Mackensen, et al. Blood 1995; 86:2699-2707; Rosenzwajg, et al., Blood1996; 87:535-544). In the presence of inflammatory cytokines theydifferentiate into macrophages activated for antigen presentation andhost defense (Munn et al., Cancer Res. 1993; 53:2603-2613; Morahan, etal. In: Heppner G H, Fulton A M, eds. Macrophages and Cancer, BocaRaton, Fla.; CRC Press, 1988:1-25). In the absence of inflammatorycytokines, monocytes differentiate under the influence of theirlineage-specific growth factor, MCSF, into a type of macrophage whichinhibits, rather than supports, T cell activation (Munn, et al. J.Immunol. 1996; 156:523-532).

The adaptive immune system must tailor the T cell repertoire so as notto respond to self antigens. The classical model (reviewed by Nossal inCell 1994; 76:229-239) holds that autoreactive T cell clones are deletedin the thymus via the process of negative selection (encounter withantigen at the immature thymocyte stage triggers apoptosis, resulting inclonal deletion). Although the thymus undoubtedly provides a major siteof negative selection, there are two difficulties with this model.First, it would seem unlikely that every developing T cell could beexposed to every self protein during its relatively brief transitthrough the thymus. Second, autoreactive T cells are empirically foundin the peripheral blood of normal, healthy hosts (Steinman Cell 1995;80:7-10). This suggests that there must exist some additional means oftailoring the T cell repertoire after the T cells have left the thymus,a process now designated peripheral tolerance. Multiple mechanisms havebeen proposed to contribute to peripheral tolerance (for recent reviewssee Steinman L. Cell 1995; 80:7-10; Mondino, et al. Proc. Natl. Acad.Sci USA 1996; 93:2245-2252; and Quill H. J. Immunol. 1996;156:1325-1327).

Most recent studies support a model in which dendritic cells are theprimary physiologic route of antigen presentation to T cells (Thomas etal. 1996). Under normal circumstances, this process is felt to occuronly in lymph nodes. Given this exclusive role for dendritic cells ininitiating immune responses, tissue macrophages represent a paradox.They are professional APCs, but they are also professional scavengers ofall manner of damaged cells and proteins, and hence take up a huge arrayof self antigens. Moreover, unlike dendritic cells, many of themconstitutively express MHC and costimulatory ligands (Azuma, et al.Nature 1993; 366:76-79) and function as APCs in vitro (Unanue, et al.Science 1987; 236:551-557), implying they are constantly prepared topresent antigen.

It is not known how they avoid provoking autoimmune responses. Onepossibility is that T cells never encounter tissue macrophages. This mayindeed be the case for naive T cells, since they are not thought tocirculate through tissues (Springer, et al. Cell 1994; 76:301-314).However, at times of injury and inflammation, many self antigensunavoidably enter the normal antigen-presentation pathway along withlegitimate foreign antigens (either because the dendritic cell has noway to discriminate between the two, or due to influx of debris fromdamaged tissues into the draining lymph nodes (Steinman 1995)).

Certain pathological conditions, such as AIDs (caused by the humanimmunodeficiency virus, HIV) and latent cytomegaloviral (CMV)infections, are extremely difficult to treat since the macrophages actas reservoirs for the viruses. Even though the cells are infected withvirus, they are not recognized as foreign. It is not known why thesecells are protected from the host's immune system.

It is therefore an object of the present invention to identifymechanisms by which tissue macrophages regulate T cell activation inorder to modulate autoimmune responses to the self-derived antigenswhich they present, especially in the context of infections withfacultative intracellular pathogens, such as HIV and CMV.

It has long been a mystery why a pregnant individual does not reject herfetus as foreign. Many theories have been proposed, and variousmechanisms suggested. Being able to understand and control thisphenomena would be of benefit both for the development of contraceptivesor aborticides, as well as in treatment of some women who are unable tocarry a fetus full term. Medawar, 1953, Symp. Soc. Exp. Biol. 7,320-338, pointed out 45 years ago that the mammalian conceptus ought tosurvive gestation because it carries and expresses paternally-inheritedpolymorphic genes that provide maternal immune responses when expressedby other tissues. The paradox presented by survival of fetal allograftshas not yet been explained in mechanistic terms despite much research onthe immunology of mammalian reproduction.

Three factors that might explain the immunological paradox of fetalsurvival are: (1) anatomic separation of mother and fetus, (2) antigenicimmaturity of the fetus and (3) immunologic “inertness” (tolerance) ofthe mother (Medawar 1953). Recently, attention has focused on the thirdpossibility based on evidence that the entire maternal T cell repertoireis transiently tolerized to paternal MHC class I alloantigens duringpregnancy (Tafuri, et al., 1995, Science 270, 630-633). However, it isnot clear how transient tolerance is imposed and maintained in theperipheral T cell repertoire during pregnancy.

It is therefor an object of the present invention to identify mechanismsby which rejection of the fetus by its mother are prevented.

It is a further object of the present invention to provide reagents andmethods for use thereof for terminating or maintaining pregnancies.

SUMMARY OF THE INVENTION

A mechanism of macrophage-induced T cell suppression is the selectiveelimination of tryptophan and/or increase in one or more tryptophanmetabolites within the local macrophage microenvironment viasimultaneous induction of the enzyme indoleamine 2,3-dioxygenase (IDO)and a tryptophan-selective transport system. Studies demonstrate thatexpression of IDO can serve as a marker of suppression of T cellactivation, and may play a significant role in allogeneic pregnancy andtherefore other types of transplantation, and that inhibitors of IDO canbe used to activate T cells and therefore enhance T cell activation whenthe T cells are suppressed by malignancy or a virus such as HIV.Inhibiting tryptophan degradation (and thereby increasing tryptophanconcentration while decreasing tryptophan metabolite concentration), orsupplementing tryptophan concentration, can therefore be used inaddition to, or in place of, inhibitors of IDO. Similarly, increasingtryptophan degradation (thereby, decreasing tryptophan concentration andincreasing tryptophan metabolite concentration), for example, byincreasing IDO concentration or IDO activity, can suppress T cells.Although described particularly with reference to IDO regulation, onecan instead manipulate local tryptophan concentrations, and/or modulatethe activity of the high affinity tryptophan transporter, and/oradminister other tryptophan degrading enzymes. Regulation can be furthermanipulated using cytokines such as macrophage colony stimulatingfactor, interferon gamma, alone or in combination with antigen or othercytokines.

Studies demonstrate that expression of IDO can serve as a marker ofsuppression of T cell activation, and plays a significant role inallogeneic pregnancy. Inhibitors of IDO can be used to activate T cellsand thereby induce rejection of a fetus. Studies show thatadministration of an inhibitor of IDO, 1-methyl-tryptophan, inducesspecific and uniform rejection of allogeneic conceptus. Embryo loss ispreceded by extensive inflammation, the appearance of monomuclear andneutrophil infiltrates, and degeneration of decidual tissues. Rejectionis T cell driven since a single paternally-inherited fetal MHC class Ialloantigen provokes embryo loss, and rejection does not occur ifmaternal lymphocytes are absent when IDO activity is inhibited or themother does not have functional T cells.

Administration of an inhibitor of IDO, 1-methyltryptophan, can causerejection of foreign tissue without toxicity. These studies provide amechanism whereby facultative intracellular pathogens are able to avoiddestruction by the host's T cells, even when the macrophages expressviral antigens on their surfaces. Inhibition of IDO should cause thehost to attack and kill the infected macrophages. In a preferredembodiment for treatment of HIV, the viral load is decreased usingstandard HIV therapy, usually over a period of three to six months. Thisis then followed by treatment with an inhibitor of IDO such as1-methyltryptophan at a dose equivalent to 1 g/kg, for a period ofbetween one day and a few weeks. For treatment of CMV infections,especially before cancer chemotherapy or bone marrow transplant, thepatient is treated with an inhibitor of IDO for a period effective forthe patient's own T cells to attack and kill any infected macrophages.Malignancies are treated with an IDO inhibitor until the tumor(s) isnecrotic or the cancer is in remission.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of thymidine incorporation (cpm) by T cells in thepresence of supplemental amino acids. Co-culture conditioned medium isselectively depleted of tryptophan. Human monocytes were allowed todifferentiate into macrophages then co-cultured with mitogen-activated Tcells (anti-CD3). Fresh PBMC were then suspended in this medium andactivated with additional anti-CD3. Replicate cultures were supplementedwith individual amino acids to the concentrations normally found inRPMI-1640. Control cultures received fresh medium. Proliferation wasmeasured by 4 hr thymidine incorporation after 72 hrs. Error bars showSD.

FIG. 2 is a graph of thymidine incorporation as a function of tryptophanconcentration (nM) showing the dose-response relationship to tryptophan.Tryptophan was titrated in co-culture conditioned medium as described inFIG. 1, and proliferation of T cells measured at 72 hrs. SD was lessthan 10% and error bars are omitted for clarity.

FIG. 3 is a graph of tryptophan concentration (micromoles) over time inhours showing the elimination kinetics of tryptophan in co-cultures.MCSF-derived macrophages were cultured for 24 hrs with autologous Tcells, with or without anti-CD3 mitogen. The medium was replaced withfresh medium, and supernatant from replicate cultures harvested at thetimes shown. The concentration of tryptophan was determined by theformaldehyde/FeCl₂ fluorescence assay. Error bars (less than 10%) areomitted for clarity.

FIGS. 4A and 4B are graphs showing inhibition of the IDO enzymeabrogates suppression. Co-cultures of MCSF-derived Mφs and T cells withanti-CD3 mitogen were supplemented either with the IDOinhibitor-1-methyl-tryptophan (FIG. 4A), or with 10× the normalconcentration of tryptophan (FIG. 4B). Proliferation was measured after72 hrs by thymidine incorporation. The level of background proliferationcontributed by macrophages is indicated by the dotted line. In FIG. 4B,macrophages were seeded at both low density (5×10⁴/well) and highdensity (2×10⁵/well). In both parcels, all differences discussed assignificant were p<0.01 by ANOVA and Tukey's.

FIG. 5 is a graph of tryptophan degradation (% total) versus interferonconcentration (U/ml) showing that CD40-L acts synergistically with IFNγto induce IDO activity. MCSF-derived macrophages were activated for 24hrs with various concentrations of recombinant IFNγ, in the presence orabsence of recombinant CD40-L trimer. At the end of the activationperiod the tryptophan remaining in the cultures was assayed as describedfor FIG. 3. SD was less than 10%.

FIG. 6 is a graph of tryptophan degradation (% total) over time (hours)for showing developmental regulation of inducible IDO activity.Monocytes were allowed to differentiate in either MCSF or GMCSF+IL-4 for5 days, then activated by sequential exposure to CD40-L and IFNγ.Following activation, the medium was replaced with fresh medium and therate of tryptophan catabolism measured over 8 hrs. SD was less than 10%.

FIGS. 7A and 7B are graphs of tryptophan uptake (pmol/10⁶ cells) overtime (min) showing inducible transport of tryptophan into macrophages.FIG. 7A shows uptake of [³H] tryptophan (200 nM) was measured for thetimes indicated, then cells were washed onto glass fiber filters andassayed by liquid scintillation counting. The graph compares freshmonocytes, MCSF-derived macrophages (day 7), and MCSF-derivedmacrophages activated for the preceding 24 hrs with IFNγ. All assayswere performed in the presence of 140 mM NaCl. FIG. 7B showssodium-dependent and sodium-independent (140 mM choline chloride) uptakefor T cells activated for 72 hrs with anti-CD3, then assayed as in FIG.7A. A logarithmic scale is used to permit comparison.

FIGS. 8A and 8B are graphs of tryptophan uptake (cpm) in the presence ofvarious competing unlabeled amino acids for dendritic cells (FIG. 8A)and MCSF macrophages (FIG. 8B) showing sodium-independent transport oftryptophan in MCSF-derived macrophages does not have the characteristicsof system L. Monocytes were allowed to differentiate for 5 days in thepresence of either GMCSF+IL-4 (dendritic cells, FIG. 8A) or MCSF(macrophages, FIG. 8B). Sodium-independent tryptophan transport wasassayed as described in FIG. 7 in the presence of various competingunlabeled amino acids (8 mM each).

FIG. 9 is a graph of percent inhibition versus concentration ofcompetitor (tryptophan or phenylalanine) showing that the tryptophantransport system in MCSF-derived macrophages displays a preferentialaffinity for tryptophan. Macrophages were cultured for 7 days in MCSFand activated during the last 24 hrs with IFNγ. Uptake of [³H]tryptophan(400 nM) was then assayed in the presence of various concentrations ofunlabeled tryptophan or phenylalanine.

FIG. 10 are HPLC analyses showing elimination of autoreactive T cells invivo is accompanied by tryptophan degradation. Antigen-transgenic micereceived autoreactive T cells by adoptive transfer, and then wereassayed for the tryptophan breakdown product kynurinine at a time whenthe T cells were being actively eliminated (day 3). Trace 1 shows HPLCanalysis for kynurinine in control serum prior to adoptive transfer.Trace 3 shows control serum spiked with authentic kynurinine (10 μM).Trace 2 shows serum from mice on day 3 after adoptive transfer of Tcells. The asterisk indicates the kynurinine peak.

FIG. 11 is a graph of autoreactive TcR-transgenic T cells (1×10⁷) overtime (days) after adoptive transfer of cells with and without inhibitor.

DETAILED DESCRIPTION OF THE INVENTION Roles of IDO and Tryptophan in TCell Activation

As monocytes differentiate into macrophages they can adopt markedlydifferent phenotypes, depending on the cytokine milieu. Macrophages maypresent antigens in an immunosuppressive, rather than immunostimulatoryfashion. Most phenotypes support T cell activation, but one, produced byexclusive exposure to the cytokine macrophage colony-stimulating factor(MCSF), suppresses T cell activation. It is proposed that macrophagesdiscriminate which antigens are “self” based on the presence or absenceof inflammation during terminal macrophage differentiation. Inflammatorycytokines such as IFNγ (Munn, et al. 1993; Munn, et al. 1996), IFNα, andIL-4 act dominantly over MCSF to prevent the development of theinhibitory macrophage phenotype. Thus, immunosuppressive macrophagesshould develop only if they differentiated in normal, uninflamedtissues. In such tissues, the only antigens encountered should beself-derived, and hence responding T cells would be presumptivelyautoreactive. This is conceptually related to proposed models ofself/non-self discrimination such as the model described by JanewayImmunol. Today 1992; 13:11-16, in which antigens in normal tissues areconsidered self, and only in settings of inflammation or infection are Tcells allowed to activate. This model has been subsequently reviewed byIbrahim et al. in Immunol. Today 1995; 16:181-186.

The data and proposed mechanisms described herein complement thesemodels by suggesting a specific mechanism by which antigens could bepresented in normal tissues in an immunosuppressive fashion. Themechanism of this suppression is via induction of the enzyme indoleamine2,3-dioxygenase (IDO), which selectively degrades the essential aminoacid tryptophan. In non-hepatic tissues, the enzyme responsible for theinitial rate-limiting step in tryptophan degradation along thekynurinine pathway is indoleamine-2,3-dioxygenase-(IDO) (Taylor, et al.FASEB J. 1991; 5:2516-2522). IDO is a subject of active current interestin the context of infectious disease (Pfefferkorn Proc. Natl. Acad. Sci.USA 1984; 81:908-912; Gupta et al. Infect. Immun. 1994; 62:2277-2284;Musso et al.; Blood 1994; 83:1408-1411; Koide et al. Infect. Immun.1994; 62:948-955; Daubener Immunol. 1995; 86:79-84; Daubener, et al.,Eur. J. Immunol. 1996; 26:487-492; Carlin, et al. J. Leuk. Biol. 1989;45:29-34; Nagineni et al. Infect. Immun. 1996; 64:4188-4196), where itis postulated to reduce the intracellular concentration of tryptophan tothe point that facultative intracellular pathogens are unable toreplicate (tryptophan is an essential amino acid for all eukaryoticparasites, and for certain simple prokaryotes). Cell lines which havemutations in the IDO gene are unable to control the replication oforganisms such as toxoplasma and chlamydia (Thomas et al. J. Immunol.1993; 150:5529-5534). Conversely, pathogen-sensitive cell linestransfected with IDO acquire the ability to inhibit proliferation ofthese organisms (Gupta et al. Infect. Immun. 1994; 62:2277-2284). IDOmay be particularly critical in defense against CNS and oculartoxoplasmosis, since glial and retinal cells show a direct correlationbetween IDO activity and control of toxoplasma (Daubener et al. Eur. J.Immunol. 1996; 26:487-492; Nagineni et al. Infect. Immun. 1996;64:4188-4196). IDO may also contribute to the pathophysiology of centralnervous system infection, autoimmune inflammation, and AIDS dementia,via both local tryptophan starvation and by generation of neurotoxictryptophan metabolites such as quinolinate (Venkateshan et al. Proc.Natl. Acad. Sci. USA 1996; 93:1636-1641; Vogelgesang et al., J.Rheumatol. 1996; 23:850-855; Sardar Neurosci. Let. 1995; 187:9-12;Alberati-Giani et al. J. Neurochem. 1996; 66:996-1004).

IDO is induced by several inflammatory mediators, including interferonsand LPS, as well as by viral infection (probably indirectly viainterferons). The most potent inducer of IDO is IFN, due to twointerferon-stimulated response elements (ISREs) in the IDO promoter(Konan et al. J. Biol. Chem. 1996; 271:19140-19145; Chon et al. J. Biol.Chem. 1996; 271:17247-17252). In addition to its effect on intracellularorganisms, IDO appears to mediate at least part of the antiproliferativeeffect of IFNγ on replicating host cells, such as virally infected cellsand tumor cells (Aune, et al., 1989 J. Clin. Invest. 84, 863-875).Mutant cell lines selected for their ability to grow in the presence ofIFNg were found to have acquired mutations in the IDO gene or itspromoter (Feng et al. Proc. Natl. Acad. Sci USA 1989; 86:7144-7148).Allogeneic tumor cells being rejected by the host immune system in vivoupregulate IDO, and this effect is mediated by IFNg (Takikawa, et al.In: Schwarcz R, ed. Kynurenine and Serotonin Pathways, New York: PlenumPress, 1991; 437-444; Yu, et al., Intl. Immunol. 1996; 8:855-865),although it is not known whether IDO plays a causal role in tumorrejection. Thus, IDO can inhibit proliferation of both intracellularpathogens and host cells.

The prior art in the field has viewed IDO solely as a means to reducethe concentration of tryptophan within the IDO-expressing cell (Taylorand Feng, 1991 FASEB J. 5:2516-2522). There has been no speculation thatIDO could function to suppress proliferation of neighboring cells, andspecifically no suggestion that it could be used to suppress T cellactivation. Based on the in vitro studies described herein, thehypothesis was formulated that cells expressing IDO would be able tosuppress T cell activation in vivo. From this hypothesis the testableprediction was formulated that inhibition of IDO enzyme activity withcompounds such as 1-methyl-tryptophan would allow enhanced T activationin vivo. Review of the existing literature suggested that IDO activitywas likely to be present in macrophage-like and dendritic-like cells inorgans of the immune system (Moffett, et al., 1994 Cell Tissue Res.278:461-469). Therefore, to test the hypothesis that IDO-expressingcells in the immune system suppressed autoreactive T cells in vivo,adoptive transfer studies were performed in which T cells which weretransgenic for a defined T cell receptor were injected into recipientmice made transgenic for the cognate target antigen. This model ofautoimmune T cell activation has been previously described by Tarazona,et al., 1996 Int. Immunol. 8:351-358. As shown in FIG. 10, the injectionof autoreactive T cells resulted in induction of IDO (measured aselevated kynurenine in serum). When IDO activity was inhibited inrecipient animals by administration of 1-methyl-tryptophan, theactivation of the autoreactive T cells was markedly enhanced (2- to3-fold over control, as shown by FIG. 11). These data thus supported thehypothesis that there existed a population of IDO-expressing cells whichsuppressed autoreactive T cells in vitro.

The in vitro data presented herein were derived using humanmonocyte-derived macrophages. For convenience, the term “macrophage” istherefore used to describe any IDO-expressing cell which suppresses Tcell activation. However, in vivo this cell could be a macrophage,dendritic cell, or other cell type with expresses IDO and suppresses Tcells.

As determined in the studies described herein, macrophages are primedduring differentiation in the presence of MCSF to undergo a massiveinduction of IDO activity in response to a synergistic combination ofsignals displayed during early T cell activation (IFNγ plus CD40ligand). In addition, these macrophages markedly increase their rate oftryptophan uptake, via induction of a previously unidentifiedhigh-affinity transport pathway. Macrophages which have differentiatedunder the influence of macrophage colony-stimulating factor (MCSF)inhibit attempted T cell activation via super-induction of theantimicrobial host-defense enzyme indoleamine 2,3-dioxygenase, whichselectively degrades the essential amino acid tryptophan.Simultaneously, these macrophages also markedly increase tryptophanuptake via induction of a novel high-affinity transport pathway. Thecombination of these two mechanisms allows macrophages of this phenotypeto effectively deplete tryptophan from the local microenvironment, thuspreventing T cell activation.

Nutrient depletion is an evolutionarily ancient strategy, and as theutility of L-asparaginase against leukemia attests it remains aneffective one. The best studied example of this type of mechanism inhumans is chelation of iron, an essential micronutrient for all dividingcells. Microbes employ this strategy against each other (deferoxamine isan example), and so do macrophages via the secretion of theiron-chelating protein lactoferrin (Baynes Adv. Exp. Med. Biol. 1994;357:133-141). Macrophages use iron depletion as a cytostatic mechanismagainst tumor cells and other dividing cells (reviewed in Weiss, et al.Immunology Today 1995; 16:495-500), and iron is rapidly sequestered fromserum and tissues by macrophages in response to infection andinflammation. Another example of regulation by nutrient depletion is theability of macrophages to alter the intracellular thiol pool in T cellsvia degradation of cysteine, resulting in thiol-mediated redoxregulation of T cell activation (Gmunder, et al. Cell. Immunol. 1990;129:32-46; Iwata, et al., J. Immunol. 1994; 152:5633-5642). Thus,tryptophan degradation is not a unique phenomenon, but rather belongs toa class of mechanisms whereby proliferation is inhibited by localdepletion of a factor essential for growth. Like macrophages themselves(Ottaviani, et al. Immunol. Today 1997; 18:169-174), the IDO gene isevolutionarily ancient. An homologous gene exists in lowerinvertebrates, and the intron-exon structure has been highly conservedthroughout 600 million years of evolution (Suzuki, et al. Biochem.Biophys. Acta 1996; 1308:41-48). This suggests that tryptophandegradation is an effective and important strategy.

A key assumption implicit in the hypothesis is that macrophages in vivoare able to create a local microenvironment in which the tryptophanconcentration is very low, despite the availability of tryptophan in thebloodstream. In theory, this should be possible because the rate oftryptophan delivery is limited by diffusion through the interstitialspace. Burke, et al, have directly tested this assumption by measuringtryptophan concentrations in tumor xenografts which had been induced toexpress IDO by IFNγ (Burke, et al. Int. J. Cancer 1995; 60:115-122).These authors found an approximately 80% reduction in tumor tryptophanin IFNγ-treated animals compared to controls, indicating that the rateof local degradation did indeed exceed the rate of delivery.

The IDO enzyme is known from published literature to be expressed insyncytiotrophoblast cells of the human placenta (Kamimura, et al., 1991Acta. Med. Okayama 45:135-139). Syncytiotrophoblasts are fetal-derivedcells which comprise the zone of contact between fetal tissues and thematernal bloodstream (and hence contact with the maternal immunesystem). The presence of IDO in placenta had been confusing toresearchers in the field, since the placenta supplies nutrients to thefetus and it seemed paradoxical that it should actively degrade anessential nutrient such as tryptophan. Based on the in vitro and in vivostudies described herein, the hypothesis was formulated that the role ofIDO in the placenta was to suppress T cell responses against thegenetically “foreign” fetus. The survival of the mammalian fetus haslong been a paradox in the field of immunology (Medawar, 1953, Symp.Soc. Exp. Biol. 7:320-338), since half of the fetus's genes arepaternally inherited, and hence should provoke an allogeneic rejectionresponse from the maternal immune system. Based on the data herein, itwas predicted that if IDO activity were inhibited in placenta the fetuswould be unable to protect itself against maternal T cells, and would berejected. As shown in Table 1, inhibition of IDO with1-methyl-tryptophan resulted in prompt, T cell-mediated rejection of allallogeneic fetuses. There was no effect of inhibitor on geneticallyidentical (syngeneic) control fetuses, demonstrating that the inhibitoritself was not toxic. This provided definitive support for the abilityof IDO to suppress T cell responses in vivo. It also showed thatIDO-expressing cells other than macrophages (in this case, trophoblasts)were able to suppress T cell responses, indicating that IDO is a broadlyapplicable mechanism of immune suppression. Specifically, based on thedata herein, it is believed that antigen-presenting cells (such asmacrophages and dendritic cells) throughout the immune system use IDO tosuppress unwanted T cell responses (e.g., to self antigens).

The fetus represents a dramatic example of a solid-tissue “allograft”which is tolerated by the maternal immune system throughout gestation.Prior to the discoveries described herein, it was uncertain whether thefetus presented the transplantation antigens needed to provokerejection, and, if it did, why the maternal immune system chose totolerate these antigens. The data in the examples reveal that the fetusdoes indeed present the relevant transplantation antigens (a differencein even a single MHC gene was sufficient to provoke rejection when IDOwas inhibited, see Table 3), and that the maternal immune system did not“choose” to tolerate the fetus, but rather was actively prevented frommounting a response by placental expression of IDO. Taken together,these observations lead to two categories of clinical applications:

Category 1: Since IDO and tryptophan catabolism represent a newmechanism of T cell suppression, the introduction of the IDO gene bytransgenesis, the use of pharmacologic inducers of IDO enzyme activity,or the direct introduction of IDO or IDO-like enzymatic activity aspurified proteins into relevant sites, can all be used for localimmunosuppression.

Category 2: Since inhibition of IDO is able to restore T cell responseswhich would otherwise be suppressed (as in the pregnancy model describedherein), pharmacologic inhibitors of the IDO enzyme could be used torestore desirable T cell responses which are normally suppressed by IDO.

With regard to Category 1 (use of IDO as an immunosuppressant), the mostdirect application is to simply inject or otherwise deliver the purifiedIDO enzyme as a local immunosuppressive agent. However, because IDOcontains a heme prosthetic group and requires a complex regeneratingsystem in order to avoid auto-oxidation in vitro (Sono, et al., 1980. J.Biol. Chem. 255:1339-1345), it may be difficult to employ the purifiedenzyme directly as a pharmacologic agent. A strategy to surmount thisdifficulty is to use purified bacterial enzymes such as indolyl-3-alkanealpha-hydroxylase, which have IDO-like tryptophan-degrading activity andwhich have been shown to function in purified form (U.S. Pat. Nos.5,723,325 and 5,244,807 to Murtfeldt, et al).

A second approach is to confer IDO enzymatic activity in atissue-specific manner by the use of IDO as a transgene under aninducible or constitutive promoter. There is currently significantinterest in developing genetically modified animals (e.g., swine) toserve as donors for solid organ xenotransplantation (Saadi and Platt1998 Life Sci. 62:365-387). Based on the data herein demonstrating therole of IDO in preventing rejection of the allogeneic fetal “allograft,”the use of IDO as a transgene (either as a germline transgene ingenetically modified animals, or delivered by transfection into humanorgans) can be applied to xenogeneic and allogeneic human organtransplantation.

Likewise, there exists a body of literature supporting the use ofgenetically modified cells to convey immunosuppressive genes intospecific anatomic sites of inflammation, e.g., into the synovial spacein arthritic joints (Jorgensen and Gay 1998. Immunol. Today 19;387-391), for purposes of local immunosuppression, or systemically forpurposes of inducing tolerance to subsequent allograft transplantation.These would be additional applications of IDO used as a transgene.

Finally, there is a well-established but poorly understood correlationbetween tryptophan metabolism and autoimmune disorders, as describedfurther in Example 8. Based on the data herein, it is proposed that IDOsuppresses autoreactive T cell activation by degrading tryptophan inlocal microenvironments. From this it follows that elevated systemictryptophan levels would make it more difficult for macrophages todeplete tryptophan locally and hence suppress autoreactive T cells;whereas lowered systemic tryptophan levels would favor suppression ofautoreactive T cells. Therefore, methods to lower systemic tryptophanlevels by pharmacologic induction of IDO, administration oftryptophan-degrading enzymes, or ex vivo depletion of plasma tryptophanby tryptophan-degrading enzymes, could be used in the treatment ofautoimmune disorders. Examples of suitable enzymes for loweringtryptophan such as indolyl-3-alkane alpha-hydroxylase, and means for exvivo treatment of plasma to lower tryptophan levels which are abnormallyelevated are described in U.S. Pat. Nos. 5,723,325 and 5,244,807 toMurtfeldt, et al.

Inhibitors of the IDO enzyme (such as 1-methyl-DL-tryptophan,β-(3-benzofuranyl)-DL-alanine, 6-nitro-L-tryptophan andβ-[3-benzo(b)thienyl]-DL-alanine), or the high affinity tryptophantransporter can be used to simulate T cell mediated immune responseswhere these would normally be suppressed by IDO. Applications includeusing these agents (systemically or locally administered) to anindividual to terminate pregnancy. Other applications includetransfection of the IDO gene (under an inducible promoter) into tissuesand cells prior to or at the time of pregnancy, to reduce thepossibility of immune-mediated rejection of the fetus. Screening oftryptophan levels and/or IDO expression can be used as indicators of Tcell activation or suppression and therefore predictors of miscarriageor spontaneous abortion.

Based on these studies and the underlying mechanism, it is possible toterminate, or help to sustain in some individuals, pregnancy. Toterminate the pregnancy one administers an effective amount of aninhibitor of IDO, or transport of tryptophan by the high affinitytryptophan transporter. For example, an inhibitor such as1-methyltryptophan at a dosage of 1 g/kg is effective to cause rejectionand killing by macrophages. The dosage may be administered one or moretimes, as required, to yield the desired effect.

Compounds which Modulate Tryptophan or Tryptophan Metabolite

A number of techniques are known for obtaining compounds which can beused to modulate tryptophan or tryptophan metabolite levels. The activecompounds can be divided into the following categories:

(1) IDO and other enzymes whose activity can be modulated to altertryptophan degradation

(2) The high affinity sodium-independent extremely selective tryptophantransporter and other transporters of tryptophan which can increase ordecrease tryptophan transport into the cell

IDO is well characterized, as discussed above. Bacterial enzymes, alsodiscussed herein, can also be used to degrade tryptophan. Tryptophantransporters are described herein and in the literature. Although thefollowing description is primarily directed towards modulation of IDOactivity, the descriptions are equally applicable for modulators of thehigh affinity tryptophan transporter, which can be regulated in the samemanner as other transporter molecules, by turning on or off transport bycompletely blocking access to the transporter, or by partially blockingaccess, for example, by binding of the transporter with a molecule whichcompetitively binds with tryptophan, or which has a higher bindingaffinity or lower dissociation constant than tryptophan. IDO is anintracellular enzyme. In order for it to affect the extracellular levelof tryptophan its substrate must be transported across the macrophagecell membrane. There are many known amino acid transport systems inmammalian cells, a number of which accept tryptophan. However, none ofthese are specific for tryptophan, so it must compete with various otheramino acids for uptake. To date, no preferential tryptophan transporterhas been described in mammalian cells. Data suggests that there mayexist an IFNγ-inducible, high-affinity, tryptophan-selective uptakesystem in MCSF-derived macrophages. It is sodium-independent, placing itin a limited category. The known sodium-independent systems aresummarized in Kakuda et al. J. Exp. Biol. 1994; 196:93-108; McGivan etal. Biochem. J. 1994; 299:321-334. In some ways the macrophage transportsystem resembles system L. However, in cross-competition studies, thetryptophan uptake system in macrophages shows a 10- to 100-fold higheraffinity for tryptophan than for the next best substrate, phenylalanine,which does not correspond to any of the known systems. System T, whichalso accepts tryptophan, has a much lower affinity than the systemobserved in macrophages (Zhou J. Biol. Chem. 1990; 265:17000-17004).

Assays for testing compounds for useful activity can be based solely oninteraction with IDO or enzymes or the transporters involved intryptophan metabolism (“the enzymes”), or alternatively, the assays canbe based on interaction with the gene sequence encoding the enzymes. Forexample, antisense which binds to the regulatory sequences, and/or tothe protein encoding sequences can be synthesized using standardoligonucleotide synthetic chemistry. The antisense can be stabilized forpharmaceutical use using standard methodology (encapsulation in aliposome or microsphere; introduction of modified nucleotides that areresistant to degradation or groups which increase resistance toendonucleases, such as phosphorothioates and methylation), then screenedinitially for alteration of enzyme activity in transfected or naturallyoccurring cells which express the enzyme, then in vivo in laboratoryanimals. Typically, the antisense would inhibit expression. However,sequences which block those sequences which “turn off” synthesis canalso be targeted

Compounds which inhibit IDO are known, as demonstrated by the examples,although not previously described for use as immunomodulators.Additional compounds which are highly selective can be obtained asfollows.

Random Generation of Enzyme or Enzyme Encoding Sequence BindingMolecules.

Molecules with a given function, catalytic or ligand-binding, can beselected for from a complex mixture of random molecules in what has beenreferred to as “in vitro genetics” (Szostak, TIBS 19:89-93, 1992). Onesynthesizes a large pool of molecules bearing random and definedsequences and subjects that complex mixture, for example, approximately10¹⁵ individual sequences in 100 μg of a 100 nucleotide RNA, to someselection and enrichment process. For example, by repeated cycles ofaffinity chromatography and PCR amplification of the molecules bound tothe ligand on the column, Ellington and Szostak (1992) estimated that 1in 10¹⁰ RNA molecules folded in such a way as to bind a given ligand.DNA molecules with such ligand-binding behavior have been isolated(Ellington and Szostak, 1992; Bock et al, 1992).

Computer Assisted Drug Design

Computer modeling technology allows visualization of thethree-dimensional atomic structure of a selected molecule and therational design of new compounds that will interact with the molecule.The three-dimensional construct typically depends on data from x-raycrystallographic analyses or NMR imaging of the selected molecule. Themolecular dynamics require force field data. The computer graphicssystems enable prediction of how a new compound will link to the targetmolecule and allow experimental manipulation of the structures of thecompound and target molecule to perfect binding specificity. Predictionof what the molecule-compound interaction will be when small changes aremade in one or both requires molecular mechanics software andcomputationally intensive computers, usually coupled with user-friendly,menu-driven interfaces between the molecular design program and theuser.

Examples of molecular modelling systems are the CHARMm and QUANTAprograms, Polygen Corporation, Waltham, Mass. CHARMm performs the energyminimization and molecular dynamics functions. QUANTA performs theconstruction, graphic modelling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive withspecific proteins, such as Rouvinen et al., 1988 Acta PharmaceuticaFennica 97, 159-166; Ripka, New Scientist 54-57 (Jun. 16, 1988);McKinlay and Rossmann, 1989 Annu. Rev. Pharmacol. Toxicol. 29, 111-122;Perry and Davies, QSAR: Quantitative Structure-Activity Relationships inDrug Design, Proceedings of the 7^(th) European Symposium on QSAR heldin Interlaken, Switzerland, Sep. 5-9, 1988, pp. 189-193 (Alan R. Liss,Inc. 1989); Lewis and Dean, 1989 Proc. R. Soc. Lond. 236, 125-140 and141-162; and, with respect to a model enzyme for nucleic acidcomponents, Askew, et al., 1989 J. Am. Chem. Soc. 111, 1082-1090. Othercomputer programs that screen and graphically depict chemicals areavailable from companies such as BioDesign, Inc., Pasadena, Calif.,Allelix, Inc, Mississauga, Ontario, Canada, and Hypercube, Inc.,Cambridge, Ontario. Although these are primarily designed forapplication to drugs specific to particular proteins, they can beadapted to design of drugs specific to regions of DNA or RNA, once thatregion is identified.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichare inhibitors or activators.

Generation of Nucleic Acid Regulators

Nucleic acid molecules containing the 5′ regulatory sequences of theenzyme genes can be used to regulate or inhibit gene expression in vivo.Vectors, including both plasmid and eukaryotic viral vectors, may beused to express a particular recombinant 5′ flanking region-geneconstruct in cells depending on the preference and judgment of theskilled practitioner (see, e.g., Sambrook et al., Chapter 16).Furthermore, a number of viral and nonviral vectors are being developedthat enable the introduction of nucleic acid sequences in vivo (see,e.g., Mulligan, 1993 Science, 260, 926-932; U.S. Pat. No. 4,980,286;U.S. Pat. No. 4,868,116; incorporated herein by reference). Nucleic acidcan be encapsulated in cationic liposomes which can be injectedintravenously into a mammal (Zhu et al., 1993 Science 261, 209-211).

The 5′ flanking sequences of the enzyme gene can also be used to inhibitthe expression of the enzyme. For example, an antisense RNA of all or aportion of the 5′ flanking region of the enzyme gene can be used toinhibit expression of the enzyme in vivo. Expression vectors (e.g.,retroviral expression vectors) are already available in the art whichcan be used to generate an antisense RNA of a selected DNA sequencewhich is expressed in a cell (see, e.g., U.S. Pat. No. 4,868,116; U.S.Pat. No. 4,980,286). Accordingly, DNA containing all or a portion of thesequence of the 5′ flanking region of the enzyme gene can be insertedinto an appropriate expression vector so that upon passage into thecell, the transcription of the inserted DNA yields an antisense RNA thatis complementary to the mRNA transcript of the enzyme gene normallyfound in the cell. This antisense RNA transcript of the inserted DNA canthen base-pair with the normal mRNA transcript found in the cell andthereby prevent the mRNA from being translated. It is of coursenecessary to select sequences of the 5′ flanking region that aredownstream from the transcriptional start sites for the enzyme gene toensure that the antisense RNA contains complementary sequences presenton the mRNA.

Antisense RNA can be generated in vitro also, and then inserted intocells. Oligonucleotides can be synthesized on an automated synthesizer(e.g., Model 8700 automated synthesizer of Milligen-Biosearch,Burlington, Mass. or ABI Model 380B). In addition, antisensedeoxyoligonucleotides have been shown to be effective in inhibiting genetranscription and viral replication (see e.g., Zamecnik et al., 1978Proc. Natl. Acad. Sci. USA 75, 280-284; Zamecnik et al., 1986 Proc.Natl. Acad. Sci., 83, 4143-4146; Wickstrom et al., 1988 Proc. Natl.Acad. Sci. USA 85, 1028-1032; Crooke, 1993 FASEB J. 7, 533-539.Inhibition of expression of a gene by antisense oligonucleotides ispossible if the antisense oligonucleotides contain modified nucleotides(see, e.g., Offensperger et. al., 1993 EMBO J. 12, 1257-1262 (in vivoinhibition of duck hepatitis B viral replication and gene expression byantisense phosphorothioate oligodeoxynucleotides); Rosenberg et al., PCTWO 93/01286 (synthesis of sulfurthioate oligonucleotides); Agrawal etal., 1988 Proc. Natl. Acad. Sci. USA 85, 7079-7083 (synthesis ofantisense oligonucleoside phosphoramidates and phosphorothioates toinhibit replication of human immunodeficiency virus-1); Sarin et al.,1989 Proc. Natl. Acad. Sci. USA 85, 7448-7451 (synthesis of antisensemethylphosphonate oligonucleotides); Shaw et al., 1991 Nucleic AcidsRes. 19, 747-750 (synthesis of 3′ exonuclease-resistant oligonucleotidescontaining 3′ terminal phosphoroamidate modifications); incorporatedherein by reference).

The sequences of the 5′ flanking region of enzyme gene can also be usedin triple helix (triplex) gene therapy. Oligonucleotides complementaryto gene promoter sequences on one of the strands of the DNA have beenshown to bind promoter and regulatory sequences to form local triplenucleic acid helices which block transcription of the gene (see, e.g.,1989 Maher et al., Science 245, 725-730; Orson et al., 1991 Nucl. AcidsRes. 19, 3435-3441; Postal et al., 1991 Proc. Natl. Acad. Sci. USA 88,8227-8231; Cooney et al., 1988 Science 241, 456-459; Young et al., 1991Proc. Natl. Acad. Sci. USA 88, 10023-10026; Duval-Valentin et al., 1992Proc. Natl. Acad. Sci. USA 89, 504-508; 1992 Blume et al., Nucl. AcidsRes. 20, 1777-1784; 1992 Grigoriev et al., J. Biol. Chem. 267,3389-3395.

Both theoretical calculations and empirical findings have been reportedwhich provide guidance for the design of oligonucleotides for use inoligonucleotide-directed triple helix formation to inhibit geneexpression. For example, oligonucleotides should generally be greaterthan 14 nucleotides in length to ensure target sequence specificity(see, e.g., Maher et al., (1989); Grigoriev et al., (1992)). Also, manycells avidly take up oligonucleotides that are less than 50 nucleotidesin length (see e.g., Orson et al., (1991); Holt et al., 1988 Mol. Cell.Biol. 8, 963-973; Wickstrom et al., 1988 Proc. Natl. Acad. Sci. USA 85,1028-1032). To reduce susceptibility to intracellular degradation, forexample by 3′ exonucleases, a free amine can be introduced to a 3′terminal hydroxyl group of oligonucleotides without loss of sequencebinding specificity (Orson et al., 1991). Furthermore, more stabletriplexes are formed if any cytosines that may be present in theoligonucleotide are methylated, and also if an intercalating agent, suchas an acridine derivative, is covalently attached to a 5′ terminalphosphate (e.g., via a pentamethylene bridge); again without loss ofsequence specificity (Maher et al., (1989); Grigoriev et al., (1992)).

Methods to produce or synthesize oligonucleotides are well known in theart. Such methods can range from standard enzymatic digestion followedby nucleotide fragment isolation (see e.g., Sambrook et al., Chapters 5,6) to purely synthetic methods, for example, by the cyanoethylphosphoramidite method using a Milligen or Beckman System 1Plus DNAsynthesizer (see also, Itakura et al., in Ann. Rev. Biochem. 1984 53,323-356 (phosphotriester and phosphite-triester methods); Narang et al.,in Methods Enzymol., 65, 610-620 (1980) (phosphotriester method).

Preparation of Enzyme Fragments

Compounds which are effective for blocking binding of the enzyme canalso consist of fragments of the enzymes, expressed recombinantly andcleaved by enzymatic digest or expressed from a sequence encoding apeptide of less than the full length enzyme. It is a routine matter tomake appropriate enzyme fragments, and test for inhibition of activityof the enzyme in the presence of the fragments. The preferred fragmentsare of human origin, in order to minimize potential immunologicalresponse. The peptides can be as short as five to eight amino acids inlength and are easily prepared by standard techniques. They can also bemodified to increase in vivo half-life, by chemical modification of theamino acids or by attachment to a carrier molecule or inert substrate.The peptides can also be conjugated to a carrier protein such as keyholelimpet hemocyanin by its N-terminal cysteine by standard procedures suchas the commercial ImJect kit from Pierce Chemicals or expressed as afusion protein, which may have increased efficacy. As noted above, thepeptides can be prepared by proteolytic cleavage of the enzymes, or,preferably, by synthetic means. These methods are known to those skilledin the art. An example is the solid phase synthesis described by J.Merrifield, 1963 J. Am. Chem. Soc. 85, 2149-2154, used in U.S. Pat. No.4,792,525, and described in U.S. Pat. No. 4,244,946, wherein a protectedalpha-amino acid is coupled to a suitable resin, to initiate synthesisof a peptide starting from the C-terminus of the peptide. Other methodsof synthesis are described in U.S. Pat. Nos. 4,305,872 and 4,316,891.These methods can be used to synthesize peptides having identicalsequence to the enzymes described herein, or substitutions or additionsof amino acids, which can be screened for activity as described above.

The peptide can also be administered as a pharmaceutically acceptableacid- or base-addition salt, formed by reaction with inorganic acidssuch as hydrochloric acid, hydrobromic acid, perchloric acid, nitricacid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organicacids such as formic acid, acetic acid, propionic acid, glycolic acid,lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,maleic acid, and fumaric acid, or by reaction with an inorganic basesuch as sodium hydroxide, ammonium hydroxide, potassium hydroxide, andorganic bases such as mono-, di-, trialkyl and aryl amines andsubstituted ethanolamines.

Peptides containing cyclopropyl amino acids, or amino acids derivatizedin a similar fashion, can also be used. These peptides retain theiroriginal activity but have increased half-lives in vivo. Methods knownfor modifying amino acids, and their use, are known to those skilled inthe art, for example, as described in U.S. Pat. No. 4,629,784 toStammer.

Pharmaceutical Compositions

Compounds which alter enzyme activity and/or tryptophan transport arepreferably administered in a pharmaceutically acceptable vehicle.Suitable pharmaceutical vehicles are known to those skilled in the art.For parenteral administration, the compound will usually be dissolved orsuspended in sterile water or saline. For enteral administration, thecompound will typically be administered in a tablet or capsule, whichmay be enteric coated, or in a formulation for controlled or sustainedrelease. Many suitable formulations are known, including polymeric orprotein microparticles encapsulating drug to be released, ointments,gels, or solutions which can be used topically or locally to administerdrug, and even patches, which provide controlled release over aprolonged period of time. These can also take the form of implants. Forexample prevention of pregnancy could be obtained by administrationintravaginally of a compound increasing tryptophan, for example, byinhibiting IDO, thereby resulting in killing of the sperm or fertilizedegg almost immediately.

Generation of Transgenic Animals for Screening

With the knowledge of the cDNA encoding the enzyme and regulatorysequences regulating expression thereof, it is possible to generatetransgenic animals, especially rodents, for testing the compounds whichcan alter enzyme expression, translation or function in a desiredmanner.

There are basically two types of animals which are useful: those notexpressing functional enzyme, and those which overexpress enzyme, eitherin those tissues which already express the protein or in those tissueswhere only low levels are naturally expressed. The animals in the firstgroup are preferably made using techniques that result in “knocking out”of the gene for enzyme, although in the preferred case this will beincomplete, either only in certain tissues, or only to a reduced amount.These animals are preferably made using a construct that includescomplementary nucleotide sequence to the enzyme gene, but does notencode functional enzyme, and is most preferably used with embryonicstem cells to create chimeras. Animals which are heterozygous for thedefective gene can also be obtained by breeding a homozygote normal withan animal which is defective in production of enzyme.

The animals in the second group are preferably made using a constructthat includes an unregulated promoter or one which is modified toincrease expression as compared with the native promoter. The regulatorysequences for the enzyme gene can be obtained using standard techniquesbased on screening of an appropriate library with the cDNA encodingenzyme. These animals are most preferably made using standardmicroinjection techniques.

These manipulations are performed by insertion of cDNA or genomic DNAinto the embryo using microinjection or other techniques known to thoseskilled in the art such as electroporation, as described below. The DNAis selected on the basis of the purpose for which it is intended: toinactivate the gene encoding an enzyme or to overexpress or express thegene encoding enzyme. The enzyme encoding gene can be modified byhomologous recombination with a DNA for a defective enzyme, such as onecontaining within the coding sequence an antibiotic marker, which canthen be used for selection purposes.

Animals suitable for transgenic experiments can be obtained fromstandard commercial sources. These include animals such as mice and ratsfor testing of genetic manipulation procedures, as well as largeranimals such as pigs, cows, sheep, goats, and other animals that havebeen genetically engineered using techniques known to those skilled inthe art. These techniques are briefly summarized below based principallyon manipulation of mice and rats. The procedures for manipulation of theembryo and for microinjection of DNA are described in detail in Hogan etal. Manipulating the mouse embryo, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1986), the teachings of which are incorporatedherein. These techniques are readily applicable to embryos of otheranimal species, and, although the success rate is lower, it isconsidered to be a routine practice to those skilled in this art.Methods for the culturing of ES cells and the subsequent production oftransgenic animals, the introduction of DNA into ES cells by a varietyof methods such as electroporation, calcium phosphate/DNA precipitation,and direct injection are described in detail in Teratocarcinomas andembryonic stem cells, a practical approach, ed. E. J. Robertson, (IRLPress 1987), the teachings of which are incorporated herein. Selectionof the desired clone of transgene-containing ES cells is accomplishedthrough one of several means. In cases involving sequence specific geneintegration, a nucleic acid sequence for recombination with the enzymegene or sequences for controlling expression thereof is co-precipitatedwith a gene encoding a marker such as neomycin resistance. Transfectionis carried out by one of several methods described in detail inLovell-Badge, in Teratocarcinomas and embryonic stem cells, a practicalapproach, ed. E. J. Robertson, (IRL Press 1987) or in Potter et al Proc.Natl. Acad. Sci. USA 81, 7161 (1984). Calcium phosphate/DNAprecipitation, direct injection, and electroporation are the preferredmethods. DNA molecules introduced into ES cells can also be integratedinto the chromosome through the process of homologous recombination,described by Capecchi, (1989).

Once the transgenic animals are identified, lines are established byconventional breeding and used as the donors for tissue removal andimplantation using standard techniques for implantation into humans.

Therapeutic Applications

The examples described herein demonstrate that tryptophan depletionand/or catabolism is the mechanism by which macrophages suppress T cellactivation, using in vitro human and in vivo transgenic mouse models;defines the developmental regulation of the IDO gene duringhematopoietic differentiation of macrophages and dendritic cells; anddefines the functional characteristics and developmental regulation ofthe tryptophan transport pathway in immunosuppressive macrophages. Thesestudies define a new and previously unsuspected role for tryptophanmetabolism to immune regulation.

Pharmaceutical compositions based on these discoveries rely on eitherincreasing tryptophan concentrations (and thereby decreasing tryptophanmetabolite concentrations) or decreasing tryptophan concentrations (andthereby increasing tryptophan metabolite concentrations). Inhibitors ofIDO increase tryptophan concentrations (and thereby decrease tryptophanmetabolite concentrations), increasing T cell activation. IDO decreasestryptophan concentrations (and thereby increases tryptophan metaboliteconcentrations), decreasing T cell activation.

Inhibitors of the IDO enzyme, such as 1-methyl-DL-tryptophan,β-(3-benzofuranyl)-DL-alanine, β-[3-benzo(b)thienyl]-DL-alanine, and6-nitro-L-tryptophan, can be used to simulate T cell mediated immuneresponses where these would normally be suppressed by IDO. Inhibitors ofother enzymes involved in tryptophan metabolism or degradation can alsobe used. Applications include using these agents (systemicallyadministered) as vaccine adjuvants to increase the immunogenicity ofadministered antigens; using the agents for immunotherapy of malignancy,including as an adjuvant for therapeutic tumor-cell vaccines, and as anadjunct to cytokine-based immunotherapy of malignancy; and using theseagents to help reverse the immunosuppressed state found in AIDS sinceIDO is known to be induced during HIV infection; co-administering IDOinhibitors with HIV vaccine to enhance the efficacy of HIV vaccinessince IDO is induced in macrophage-lineage cells by HIV infection; andgenerally for any setting in which IDO mediates unwanted suppression ofa desired immune response.

Alternatively, recombinant IDO can be used as a means of systemic orlocal T cell suppression. Applications include transfection of the IDOgene (under an inducible promoter) into tissues and cells prior toallotransplantation, in order to allow them to protect themselves fromrejection by the host, for example, by retroviral or other transfer ofthe IDO gene into allogeneic solid-organ grafts (kidney, etc.); bytransgenic expression of the gene in porcine or other animals designedas donors for xeno-transplantation, to prevent rejection; or byretroviral or other transfer into cell preparations intended fortransplantation (eg, pancreatic islet cells for therapy of diabetesmellitus).

Applications also include using these agents (systemically or locallyadministered) to an individual to terminate pregnancy. Otherapplications include transfection of the IDO gene (under an induciblepromoter) into tissues and cells prior to or at the time of pregnancy,to reduce the possibility of immune-mediated rejection of the fetus. Toterminate the pregnancy one administers an effective amount of aninhibitor of IDO, or transport of tryptophan by the high affinitytryptophan transporter. For example, an inhibitor such as1-methyltryptophan at a dosage of 1 g/kg is effective to cause rejectionand killing by macrophages. The dosage may be administered one or moretimes, as required, to yield the desired effect.

Screening of tryptophan levels and/or IDO expression can be used asindicators of T cell activation or suppression. Screening of tryptophanlevels and/or IDO expression can be used as indicators of T cellactivation or suppression and therefore predictors of miscarriage orspontaneous abortion. Testing for IDO expression tumor biopsies can beused to determine the suitability of the cancer for IDO-targetedtherapy.

The present invention will be further understood by reference to thefollowing non-limiting examples.

Example 1 Macrophage-Induced Suppression

As described in Munn, et al. J. Immunol. 1996; 156:523-532, a number ofpossible mechanisms for macrophage-induced suppression were initiallyexamined. None of those tested, including the fas/fas-L system, nitricoxide, free radicals, prostaglandins, and inhibitory cytokines, couldaccount for the phenomenon. Studies demonstrated the following regardingmacrophage inhibition of T cell proliferation:

-   (1) MCSF-derived macrophages inhibit the mitogen-induced    proliferation of T cells, and this is a dominant effect—i.e., it is    not rescued by adding non-inhibitory APCs.-   (2) The T cells initiate early activation, as assessed by    activation-related G1 phase genes (IL-2 receptor, cdc2, cyclin A),    but arrest prior to the first G1/S boundary. This arrest is not a    stable condition, and most of the lymphocytes progressively die off.-   (3) The suppressive effect was developmentally regulated by MCSF and    IFNγ. It was not present in fresh monocytes, emerged progressively    over 4-5 days of differentiation in MCSF, and was prevented if the    monocytes were exposed to IFNγ (even transiently) prior to    differentiation. IFNγ has a dual action in macrophage    differentiation. If monocytes are exposed to IFNγ prior to    differentiation it functions as a developmental regulator, and the    monocytes will not subsequently acquire the suppressive    phenotype—even in the presence of MCSF. However, if macrophages are    exposed to IFNγ after differentiation, it causes activation but does    not alter the MCSF-induced phenotype. Thus, the nature of the    response to IFNγ is determined by the developmental state of the    macrophage.-   (4) There was no evidence for participation of nitric oxide,    reactive oxygen species, TNF, TGFβ, prostaglandins, IL-1R    antagonist, or IL-10. The mechanism of inhibition appeared contact    dependent. Inhibition could not be reproduced by conditional medium    (up to 50% v/v), and was abrogated when T cells were separated from    macrophages by a semipermeable membrane. The contact-dependence is    understandable in light of the data indicating a requirement for    CD40/CD40L in order to produce full activation of tryptophan    degradation.

Example 2 Tryptophan Degradation by MCSF-Derived Macrophages

Medium in macrophage-lymphocyte co-cultures is selectively depleted oftryptophan. Conditioned medium was generated from co-cultures ofmacrophages plus T cells activated with mitogen (two different mitogens,anti-CD3 antibody and SEB, gave equivalent results; the data presentedare from anti-CD3). Fresh lymphocytes were suspended in conditionedmedium and activated with additional mitogen. Care was taken to excludeall traces of fresh medium.

As shown in FIG. 1, conditioned medium completely failed to support Tcell proliferation (less than 1% of the proliferation in fresh medium).In contrast, control conditioned media from macrophages alone, fromco-cultures of macrophages and T cells without mitogen, and from T cellsactivated with fresh monocytes instead of macrophages, all supported Tcell proliferation comparably to fresh medium (90-140%) of control,n=3-4 in each group, p=NS by ANOVA, see Munn et al. J. Immunol. 1996;156:523-532 for representative data.

Selective add-back studies showed that the deficiency lay in anessential amino acid. Supplementation with individual amino acids(FIG. 1) showed that tryptophan was depleted in conditioned medium. Theaddition of tryptophan to conditioned medium fully restored T cellproliferation, indicating that tryptophan was the only deficientcomponent. Consistent with this, HPLC analysis of conditioned mediashowed that the levels of other essential amino acids were similar tofresh medium (some concentrations were higher, presumably due to proteincatabolism).

As shown in FIG. 2, titration of reagent tryptophan in conditionedmedium gave a half-maximal concentration of approximately 500 nM for Tcell proliferation (compared to a normal level in RPMI of 25 μM). Thesestudies also showed that as little as 50 nM was sufficient to initiatelow but readily detectable T cell proliferation, indicating thattryptophan had been reduced below this range. Consistent with this,direct measurement of tryptophan in conditioned medium was below thedetection limit for our assay, which was 50 nM.

Depletion of tryptophan is rapid. Macrophages were co-cultured with Tcells and mitogen for 24 hrs to allow up-regulation of thetryptophan-depletion pathway, then fresh medium was added and thekinetics of elimination measured. As shown in FIG. 3, tryptophan waseliminated by first-order kinetics with a half-life of approximately 2hours. The initial rate of elimination (when tryptophan was notlimiting) was up to 20,000 pmol/10⁶ cells/hr. By way of comparison,fresh monocytes stimulated with IFNγ (a standard model of IDO induction)degraded tryptophan at approximately 200 pmol/10⁶ cells/hr, which isconsistent with the literature. This rapid rate of tryptophan depletioncould not be attributed to consumption by cellular metabolism. Despitetheir high metabolic activity, macrophages alone depleted tryptophan ata barely detectable rate (less than 5% of the activated rate, FIG. 3).It also did not reflect sequestration of free tryptophan withinmacrophages, since the preliminary validation studies had shown thattryptophan was undetectable in culture supernatants whether macrophageswere intact or lysed prior to performing the assay.

Example 3 Inhibition of Tryptophan Degradation by MCSF-DerivedMacrophages Prevents Suppression of T Cells

Inhibition of tryptophan degradation prevents Macrophage-mediatedsuppression of T cells. The data predicts that pharmacologic inhibitorsof IDO should prevent suppression. One potent inhibitor of IDO is the1-methyl derivative of tryptophan (Cady, et al. Arch. Biochem. Biophys.1991; 291:326-333). As shown in FIG. 4A, the presence of1-methyl-tryptophan effectively prevented macrophages from suppressing Tcell activation. These data were selected in order to clearlydemonstrate that suppression can be abrogated, but in most experimentsthe effect of 1-methyl-tryptophan has been less complete (typically59-60% reversal of suppression). This was not unexpected, given theextremely high level of enzyme activity and the fact that1-methyl-tryptophan is a competitive, rather than an irreversible,inhibitor. In order to fully abrogate macrophage-mediated suppression itwill be necessary to simultaneously block the induction of the enzymeand to pharmacologically inhibit its activity.

Macrophages can overcome high concentrations of tryptophan. A secondtest of the mechanism was to supplement the culture medium withadditional tryptophan to reverse the inhibitory effect of IDO.Tryptophan supplementation alone was never as effective as1-methyl-tryptophan, and sometimes was entirely ineffective, and asshown in FIG. 4B, appears to be related to the seeding density ofmacrophages and T cells. At low densities, supplemental tryptophaneffectively reversed suppression, but at high densities the macrophagesremained able to suppress. The effect of seeding density is believed toreflect the limited ability of diffusion to deliver tryptophan to the Tcells and macrophages.

Example 4 Effect of Cytokines on Macrophage Tryptophan Degradation

Macrophage tryptophan degradation is synergistically induced by signalsof early T cell activation. The rapid depletion of tryptophan bymacrophages was not induced by resting T cells, but occurred only whenthe T cells attempted to activate (cf. FIG. 3). This implied that themacrophages were detecting some sign of T cell activation whichtriggered tryptophan degradation. In light of the studies implicatingIFNγ as a major regulator of the IDO gene, it was predicted that Tcell-derived IFNγ would be the signal for tryptophan degradation.However, when dose-response titration was performed of recombinant IFNγon MCSF-derived macrophages, it was found that IFNγ concentrations of100 U/ml or more were required for maximal induction, and even then thelevel of enzyme activity did not reach those achieved by co-culture withactivated T cells.

CD40L is upregulated early in T cell activation and it is known to actsynergistically with IFNγ in signaling macrophages for other functions.The response of MCSF-derived macrophages to IFNγ in the presence orabsence of recombinant CD40L (prepared as a homo-trimer in order tosignal via CD40 in soluble form, gift of Bill Fanslow, ImmunexCorporation) was compared. The addition of CD40L alone had little effecton enzyme activity, but as shown in FIG. 5 it displayed marked synergywhen combined with IFNγ. The effect of CD40L was to shift thedose-response curve of IFNγ one to two orders of magnitude, so thatsignificant response now occurred at IFNγ concentrations as low as 1U/ml. Thus, two signals normally delivered by T cells to APC early inthe course of activation combined to induce tryptophan degradation inMCSF-derived Mφs.

The responsiveness of tryptophan metabolism to IFNγ and CD40L isdevelopmentally regulated. It was then determined if allmonocyte-derived cells degraded tryptophan in response to these signals,or whether it was specifically associated with the MCSF phenotype. Togenerate a non-suppressive phenotype for comparison, monocytes werecultured for 5 days GMCSF+IL-4, which produces cells that closelyresemble tissue dendritic cells, and which are potent activators of Tcells. For clarity, these cells are referred to as dendritic cells andMCSF-derived cells as macrophages. As shown in FIG. 6, dendritic cellsshowed minimal tryptophan degradation in response to IFNγ and CD40L, incontrast to MCSF-derived macrophages. Thus, the response to IFNγ was notsimply a feature of differentiated monocytes, but was regulated by thecytokine milieu present during terminal differentiation. Interestingly,when the cytokines were added sequentially, the effect of CD40L ondendritic cells was to further inhibit any residual induction oftryptophan degradation in response to IFNγ, exactly the opposite of itseffect on macrophages.

Example 5 The Tryptophan Transport System in Macrophages

Transport of tryptophan into Macrophages is developmentally regulated.Whether the upregulation of tryptophan degradation was accompanied byincreased or decreased tryptophan transport into macrophages was thendetermined. As shown in FIG. 7A, following differentiation MCSF-derivedmacrophages showed a markedly increased ability to take up tryptophan,10 to 20-fold greater than fresh monocytes, which was further induced byactivation with IFNγ.

Tryptophan transport in MCSF-derived Macrophages shows a preferentialaffinity for tryptophan. To characterize the tryptophan transportsystem, whether uptake was dependent on the sodium gradient was thendetermined. Preliminary studies had shown that tryptophan transport infresh monocytes was sodium dependent (less than 10% of transport wassodium-independent). However, as shown in FIG. 7B, the majority of thetryptophan transport in macrophages was independent of sodium. Thissodium-independent system was then characterized using cross-competitionstudies. As expected, T cells showed a pattern consistent with system L,with tryptophan uptake being inhibited by aromatic and neutral aminoacids, but not lysine or arginine (FIG. 8A). In contrast, however,tryptophan uptake by MCSF macrophages (FIG. 8B) was only partiallyinhibited by neutral amino acids, and even phenylalanine could not fullyblock it.

As shown in FIG. 9, titration studies indicated that phenylalanine (thebest competitor) was 10- to 100-fold less effective than tryptophan incompeting for uptake, based on relative IC₅₀. Tyrosine, leucine, and thesystem L-specific substrate 2-amino-2-norborane-carboxylic acid (BCH)were even less effective competitors. Significantly, when system L wasinhibited by 8 mM BCH, there still remained 30-50% of the totaltryptophan transport available (depending on the experiment). Takentogether these data suggested that a high-affinity, non-system Ltransport pathway might be present.

Example 6 In Vivo Studies of the Role of IDO and Tryptophan Degradationon Activation of T Cells

Peripheral deletion of autoreactive T cells in vivo is accompanied byevidence of tryptophan degradation. Whether there was evidence that IDOwas involved in peripheral tolerance in vivo was then assessed. SinceIDO activity at the single cell level cannot be measured in vivo, amodel in which a large number of autoreactive T cells underwentsynchronous activation and deletion was needed. This would allow one tofollow the kynurinine breakdown products released systemically when IDOdegrades tryptophan (Meyer, et al. J. Lab. Clin. Med. 1995;126:530-540). Dr. Mellor had developed such a model by creating micetransgenic for a T cell receptor recognizing the H-2K^(b) (MHC class I)antigen. When these T cells are adoptively transferred into a syngeneicanimal made transgenic for the target antigen, they become functionallyautoreactive. As described in Tarazona, et al. Intl. Immunol. 1996;8:351-358, these autoreactive T cells undergo a transient period ofactivation, but then are rapidly eliminated (day 4-5) without causingdisease. This occurs despite the fact that they are not subject torejection by the host (being syngeneic), and are eliminated only inanimals where they encounter their antigen. This therefore represents auseful model of synchronous deletion of autoreactive T cells.

Splenocytes from T cell receptor-transgenic mice (4×10⁷ cells) wereadoptively transferred by tail vein injection into antigen-transgenicmice. On day 3 (at the beginning of the rapid expansion phase), therecipient animals were sacrificed and serum was obtained. Pre-transfersera were used as controls. As shown in FIG. 10, mice which had receivedautoreactive T cells showed a marked elevation in serum kynurininecompared to controls. Thus, the expansion and elimination ofautoreactive T cells in vivo was accompanied by evidence of significanttryptophan catabolism, consistent with activation of IDO.

Whether in vivo inhibition of the IDO enzyme with 1-methyl tryptophan(as described in vitro in FIG. 4A) would alter the kinetics ofactivation or elimination of autoreactive T cells in the mouse model wasthen measured. As shown in FIG. 11, continuous infusion of 1-methyltryptophan (via subcutaneously implanted timed-release pellets, 1000mg/kg/day) resulted in significantly enhanced activation of theadoptively transferred T cells. These results strongly support a rolefor IDO in suppressing T cell activation in vivo.

Example 7 MCSF and Tissue Macrophages

Clinically, there exists an ill-defined association between alteredtryptophan metabolism and autoimmune disorders. Patients receivingL-5-hydroxytryptophan for neurologic disorders experienced a highfrequency of a scleroderma-like illness (Sternberg et al. N. Engl. J.Med. 1980; 303:782-787). Individuals who ingested certain preparationsof L-tryptophan contaminated with toxic tryptophan derivatives (1-1′ethylidenebis-[tryptophan]) experienced the eosinophilia-myalgiasyndrome (Belongia et al. N.E. Engl. J. Med. 1990; 323:357-365; Mayenoet al. Science 1990; 250:1707-1708), which included excessive T cellactivation. There is even a case report of simple dietarysupplementation with tryptophan leading to multiple autoimmune disorders(Morgan, et al. Br. J. Dermatol. 1993; 128:581-583).

The osteopetrotic (op/op) mouse lacks functional MCSF, and has aselective deficiency in specific sublets of tissue macrophages(Cecchini, et al. Development 1994; 120:1357-1372). It has normaldendritic cell development and normal T cell responses to foreignantigens (Begg et al. J. Exp. Med. 1993; 177:237-242) indicating thatthese functions are not MCSF-dependent. Interestingly, in onewell-established model of spontaneous autoimmunity, the NOD mouse, themacrophages have been shown to have a defective proliferative responseto MCSF, and fail to undergo MCSF-induced terminal differentiation(Serreze, et al. J. Immunol. 1993; 150:2534-2543). This led to speculatethat autoimmune NOD mice lack a specific, MCSF-induced tolerogenicmacrophage subset. The in vitro studies show that MCSF is a growthfactor for both immunosuppressive and inflammatory macrophages, and thatwhen inflammatory cytokines are present they act dominantly over MCSF.Thus, in the presence of dysregulated inflammatory cytokines, theaddition of MCSF would be expected to exacerbate rather than reduce thedisorder (Moore, et al. J. Immunol. 1996; 157:433-440).

Based on the preliminary data, it was proposed that IFNγ induces IDO incertain macrophage and thereby triggers T cell suppression. This is asomewhat surprising role for IFNγ, which is generally consideredpro-inflammatory rather than immunoregulatory. However, mice withtargeted disruptions of the gene for either IFNγ (Krakowski, et al. Eur.J. Immunol. 1996; 26:1641-1646) or the IFNγ receptor (Willenborg, et al.J. Immunol. 1996; 157:3223-3227) demonstrate an unexpectedhyper-susceptibility to experimental autoimmune encephalomyelitis (EAE).Mice normally resistant to this induced autoimmune disorder are renderedsusceptible in the absence of IFNγ, and the disease is fatal anduncontrolled. Adoptive transfer studies showed that the requirement forIFNγ lay with an IFNγ-responsive regulatory cell, not with theautoreactive T cells. In contrast, these mice fail to mount anappropriate inflammatory response to intracellular pathogens (Dalton, etal. Science 1993; 259:1739-1742; Kamijo, et al. J. Exp. Med. 1993;178:1435-1440). Thus, IFNγ is pro-inflammatory in infectious disease,but is immunoregulatory in at least one model of autoimmunity. This isconsistent with the hypothesis that some macrophages respond to IFNγ asa signal to inhibit T cell activation.

The following examples demonstrate that 1-methyl-L-tryptophan, aninhibitor of L-tryptophan degradation, administered during murinepregnancy induces loss of allogeneic (mother and fetus are geneticallydifferent) conceptus 2-3 days after blastocyst implantation in uterus.Syngeneic conceptus are not affected by this treatment.

Example 8 The mIDO Gene is Transcribed in Epididymis and in ConceptusDuring Pregnancy

RT-PCR analyses on RNA extracted from a panel of mouse tissues wasconducted to examine the extent of mIDO gene transcription in mice. mIDOtranscripts were detected in RNA samples from mouse epididymis but notfrom muscle, heart, bone-marrow, spleen, peripheral lymph nodes, kidney,liver, brain, intestine or lung. PCR products were generated by RT-PCRamplification from RNA samples prepared from pooled syngeneic (s) orallogeneic (a) conceptus. RT-PCR amplification of murine α-actintranscripts was conducted on each RNA sample to verify RNA integrity.Methods: Female CBA mice were mated with syngeneic or allogeneic (B6)male mice. Females were inspected daily (am) for vaginal plugs; themorning plugs were detected was taken as 0.5 dpc (days post coitus). Allconceptus dissected from each pregnant female were pooled, snap frozenon liquid nitrogen and used or prepare total RNA using standardprocedures by homogenization in RNA-STAT 60 solution (Tel-TestB Inc.).Transcripts of the murine IDO gene (Habara-Ohkubo, et al., 1991 Gene105:221-227) were detected by RT-PCR using forward(GTACATCACCATGGCGTATG, SEQ ID NO:1) and reverse (GCTTTCGTCAAGTCTTCATTG,SEQ ID NO:2) oligonucleotide primers which generated PCR products of theexpected size (740 bp). RT-PCR conditions used were 48° C., 45 min./94°C., 2 min. (1 cycle); 94° C., 30 sec./58° C., 1 min./68° C., 2 min. (40cycles); 68° C., 5 min. (1 cycle). PCR products were fractionated on a1.5% agarose/TBE gel containing ethidium, bromide and were visualized byUV fluorescence and images of gels were recorded as a digital bitmapsusing a high definition digital fluorescence and images of gels wererecorded as a digital bitmaps using a high definition digital camera.RT-PCR amplification of the murine α-actin gene (480 bp) was performedin parallel to verify RNA integrity.

In all cases, PCR products from the α-actin gene were detectedindicating that RNA samples were not degraded.

IDO gene expression has been described in human placental trophoblastcells. To examine whether IDO transcription occurs during murinepregnancy, RNA samples were prepared from uterus and conceptus ofpregnant mice at various stages of gestation. Female mice (CBA) weremated to syngeneic (CBA) or allogeneic C57BL/6 (B6) male mice andinspected daily for vaginal plugs. The morning the plug was detected wastaken as 0.5 days post coitus (dpc). RNA samples were prepared fromdissected components of conceptus; embryos, decidua (extra-embryonictrophoblast plus maternal uterus) and from uterus tissues. mIDO genetranscripts were detected in conceptus, but not uterus from syngeneicand allogeneic conceptus at early (7.5 dpc, 9.5 dpc) gestation times.mIDO transcripts were not detected in embryo RNA at 9.5 dpc suggestingthat decidual tissues were the site of IDO expression in the conceptus.At later gestation times (10.5 and 13.5 dpc) mIDO transcripts weredetected in allogeneic conceptus but not in syngeneic conceptus. RNA wasprepared from decidual tissues dissected separately from embryos. mIDOtranscripts were not detected in RNA from embryos at 10.5 or 13.5 dpc.From these data it was concluded that mIDO transcription occurs indecidual tissues of all conceptus from early gestation times (7.5-9.5dpc).

Example 9 Loss of Allogeneic Conceptus Occurs in the Presence of anInhibitor of IDO Enzyme Activity

A study was conducted to test whether IDO enzyme activity contributes tosurvival of fetal allografts by treating pregnant female mice with1-methyl-L-tryptophan, an inhibitor of IDO enzyme function. Female (CBA)mice were mated with syngeneic (CBA) or allogeneic (B6) male mice. On4.5 days post coitus (“dpc”) two slow-release pellets impregnated with1-methyl-L-tryptophan (1 g/kg/day) were surgically implanted under thedorsal skin of pregnant mice. Control mice were treated with pellets notimpregnated with 1-methyl-L-tryptophan (placebo groups). After surgery,mice were sacrificed and their uterus examined macroscopically andmicroscopically at various stages of gestation.

Based on comparison with extensive breeding records from the MCGTransgenic Unit fecundity rates for mouse colonies bred at MCG are 6.8(CBA×CBA) and 6.4 (CBA×B6) pups per female at parturition. The sameprocedure was used to assess embryo survival and development in themating combinations presented in Table 2. A large cohort of untreatedfemale CBA mice mated with GK transgenic male mice were examined and areincluded in Table 1 to show that the mean number of conceptus (atparturition) is identical to the [CBA×B6] mating combination.

Results are summarized in Table 1.

TABLE 1 Inhibition of IDO activity induces rejection of allogeneicconceptus Mating Mean No. Genotype Gestation conceptus/♀; conceptusStage (No. ♀ treated) Appearance of ♀ × ♂ (dpc) Inhibitor PlaceboInhibitor Placebo CBA × CBA 6.5-8.5 7.3 (3) 7.5 (2) normal normal 15.5 6.7 (6) 6.5 (6) normal normal CBA × B6 6.5 7.3 (3) 8.5 (2) normal normal7.5 4.5* (6)  8.0 (4) majority normal inflamed 8.5/9.5 0.5* (17) 7.3 (6)all normal inflamed 11.5-15.5 0* (7) 5.8 (4) — normal *p < 0.05 by ANOVAcompared to placebo controls at each time point

Treatment with 1-methyl-L-tryptophan had a profound affect ondevelopment of allogeneic conceptus. Syngeneic and allogeneic conceptusin mice treated with 1-methyl-L-tryptophan were found in normal numbers,were all healthy and were at the appropriate stage of fetal developmentat 6.5 dpc. Examinations conducted at 7.5 dpc, however, revealedstriking differences in the numbers and appearances of conceptus. Theaverage number of allogeneic conceptus+inhibitor was reduced to 4.5 perfemale at 7.5 dpc and declined to <2 at 8.5/9.5 dpc. No conceptus werepresent on any mice carrying allogeneic conceptus and treated with 1IDOinhibitor after 9.5 dpc. Normal numbers of healthy allogeneic (−IDOinhibitor) and syngeneic (±IDO inhibitor) conceptus were present incontrol groups. Methods. Mice were mated, treated with IDO inhibitor anddissected at gestation times indicated. Tissues were prepared forsectioning by fixing them in 4% paraformaldehyde. Serial sections (5 μm)were prepared using a microtome and were stained with hematoxylin andeosin before microscopic examination.

On gross examination significant inflammation and excessive maternalblood surrounded the majority of 7.5 dpc allogeneic conceptus in micetreated with IDO inhibitor. In contrast, maternal blood was localizednormally near the ectoplacental cone in all 7.5 dpc syngeneic conceptustreated with IDO inhibitor; their appearance was indistinguishable fromthat if syngeneic and allogeneic conceptus dissected from mothers inplacebo groups.

On histological examination increased numbers of enlarged blood vesselswithin the decidual region were observed in 7.5 dpc allogeneic conceptuswhen IDO activity was blocked. However, allogeneic 7.5 dpc embryosappeared intact and at the expected developmental stage. One day later(8.5 dpc) allogeneic conceptus (+IDO inhibitor) were grossly abnormal.Extensive mixed mononuclear and neutrophil infiltrates were evident inevery conceptus and were accompanied by extensive tissue degeneration inthe decidual region. Grossly abnormal embryos were present butdevelopmentally retarded. At 9.5 dpc no allogeneic embryos were presentin mice treated with IDO inhibitor and decidual swellings were notapparent; some cellular debris was detected within the uterine lumen.

These data reveal that blocking IDO activity during pregnancy has aprofound and specific effect on development of allogeneic conceptus,leading to loss of all embryos by mid-gestation. Embryo loss was notcaused by surgical manipulations to implant pellets at 4.5 dpc. Thisdata shows that blocking IDO activity compromises development ofconceptus only when there is a genetic difference between mother andembryos. This implies that functional IDO enzyme activity is essentialto protect allogeneic conceptus during early gestation and that thematernal immune system is involved in processes that lead to loss ofallogeneic conceptus.

Example 10 Maternal Lymphocytes Provoke Loss of Allogeneic Conceptuswhen IDO Activity is Blocked

The contribution of maternal lymphocytes to experimentally induced lossof allogeneic conceptus in mice treated with 1-methyl-L-tryptophan wasassessed using females carrying the RAG-1 induced mutation (RAG-1−/−)which prevents development of T or B cells (Li, 1982, Cancer 50,2066-2073). As before, female mice carrying disrupted RAG-1 genes (onthe DBA background) were mated with B6 males and treated with1-methyl-L-tryptophan (4.5 cpd). Pregnancies were continued until 11.5dpc when females were sacrificed to assess the number of conceptus.Normal numbers of healthy conceptus were found in all females examined(Table 2, first line).

TABLE 2 Maternal lymphocytes and a single MHC I alloantigen induceembryo rejection Mating Genotype No. IDO No. ♀ examined Mean (♀ × ♂)Inhibitor (dpc) conceptus/♀ CBA [RAG-1−/−] × B6 yes 5 (11.5) 7.8 CBA[RAG-1−/−] × B6 yes 2 7 (20.5, post partum) (pups born) CBA × GK* yes 4(11.5) 0 CBA × GK* no 21 (11.5)  6.4 *GK mice are H-2K^(b)-transgenicmice made on the CBA background.

This data demonstrates that material lymphocytes are essentialparticipants in events that precede loss of allogeneic conceptus whenIDO activity is blocked. The most likely explanation is that maternal Tcells are responsible for initiating mediating processes that lead toloss of allogeneic conceptus when IDO activity is blocked. This premiseis based on the assumption that conceptus, like tissue allografts,provoke T cell responses. Although B cells, via antibody production, cancontribute to allograft rejection, it is unlikely that antibodiesagainst fetal alloantigens mediate rapid loss of conceptus a few daysafter implantation, especially as virgin females were used in theseexperiments.

Example 11 A Single MHC 1 Difference Provokes Loss of AllogeneicConceptus

B6 and CBA mice differ at a large number of genetic loci including theentire MHC region (MHC I and MHC II) as well as at multiple minorhistocompatibility (miHC) loci. To determine whether a singlepolymorphic MHC molecule could provoke fetal loss, CBA mice were matedwith (GK) transgenic male mice carrying a recombinant H-2K^(b) (GK)transgene; a promoter from the human HLA-G gene drivesH-2K^(b)-expression at high level in murine trophoblast (Zhou, et al.1998 J. Reprod. Immunol. 40, 47-62). GK mice were made on the inbred CBAstrain background and, hence, differ from CBA mice only because ofH-2K^(b) expressed by the GK transgene. Normal size litters are borne byCBA females after mating with GK transgenic male mice (Table 2).Pregnant mice were treated with 1-methyl-L-tryptophan and examined at11.5 dpc, as before, to assess effects on fetal development (Table 2).No conceptus, nor any resorbed decidua, were observed when mice wereexamined on 11.5 dpc, although, in each mouse, the uterus was distendedindicating that pregnancy had terminated prematurely.

This result is important for two reasons. First, a singlepaternally-inherited MHC I difference between mother and conceptus isenough to provoke fetal loss when IDO activity is blocked. Second, iteliminates the possibility that fetal loss occurs after CBA [×B6]matings because of an inherent genetic predisposition to fetal loss inthis mating combination.

This result supports the view that maternal T cells, not B cells, areresponsible for fetal loss since MHC alloantigens provoke strong T cellresponses but weak B cell responses. If true, this implies that maternalCD8⁺ T cells recognizing native H-2K^(b) molecules expressed by fetalcells may be responsible for provoking fetal loss.

In summary, these data demonstrate that the IDO enzyme inhibitor1-methyl-tryptophan has a profound and specific abortifacient effect onall allogeneic conceptus prior to 9.5 dpc. Fetal abortion is not due tonon-specific toxic affects of the drug itself as syngeneic conceptusdevelop normally in females exposed to 1-methyl-tryptophan as doallogeneic conceptus in the absence of maternal lymphocytes.Alternatively, embryonic development may be affected by substancesproduced when 1-methyl-tryptophan is metabolized by cells expressingIDO. However, this is unlikely to explain the abortifacient effect of1-methyl-tryptophan since the IDO gene is expressed in syngeneic andallogeneic conceptus early in gestation during the period when embryorejection occurs (7.5-9.5 dpc). One therefore concludes that theabortifacient effect of 1-methyl-tryptophan arises because it inhibitsIDO enzyme activity. Decidual cells expressing IDO normally establish aprotective barrier that prevents maternal T cell mediated rejection ofallogeneic conceptus.

Example 12 Inhibition of Tumor Growth by Administration of IDO Inhibitor

In light of the data herein showing that the allogeneic fetus is able toprotect itself against attack by the maternal immune system throughexpression of IDO, and given the expression of IDO in tumors as citedabove, it was tested whether a similar mechanism allowed tumor cells toevade immune attack. The MB49 epithelial carcinoma model was chosen forproof of concept because it is an aggressive, lethal malignancy wheninnoculated into syngeneic hosts, but it can be rendered susceptible toimmune attack if the host's immune system is suitably activated (Chen,et al. 1997 J. Immunol. 159, 351-359). Tumor-bearing hosts were treatedwith the IDO inhibitor 1-methyl-tryptophan. MB49 tumor cells (1×10⁶)were injected subcutaneously into syngeneic C57/B16 hosts. Pelletscontaining 1-methyl-tryptophan (0.9 mg/hr, 7-day release) were implantedat the time of tumor cell inoculation. By day 10, all animals hadevidence of initial tumor formation (palpable mass). By day 15, controlanimals were visibly ill and the experiment was terminated. Animals weresacrificed on day 11-15 for histologic examination.

As shown in Table 3, administration of 1-methyl-tryptophan significantlyreduced tumor growth in immunocompetent, syngeneic hosts, compared tovehicle control. Two important points are noted: First, no manipulationof the immune system other than inhibition of IDO was required foranti-tumor effect—i.e., the tumor itself became immunogenic once thebarrier of IDO was removed. Second, the observed responses continued atleast 4-7 days after the period of inhibitor administration (days 1-7),suggesting that an initial priming period was sufficient to generate asustained anti-tumor immune response.

TABLE 3 Anti-tumor effects of 1-methyl-tryptophan administration.control group 1-methyl-tryptophan Outcome measure (n = 7) group (n = 7)complete responses 0 1 partial responses 0 6 size of residual 2-2.5 cm 1cm (necrotic) tumor histology (day 15) viable tumor, minor apoptosis,necrosis, infiltration of CD4+ T cells and hemorrhage, with occasionalCD8+, extensive infiltrate of scattered macrophages CD4+ and CD8+ Tcells, extensive infiltration of macrophages visible metastasesextensive local metastases, none with gross evidence of intraperitonealand visceral metastases performance status decreased activity, failurenormal to groom, ill appearance

Modifications and variations of the methods and compositions describedherein will be obvious to those skilled in the art and are intended tobe encompassed by the claims.

1. A method of supplementing standard antiretroviral HIV therapy, themethod comprising administering an effective amount of a compositioncomprising 1 methyl-tryptophan to an individual already undergoing astandard antiretroviral HIV therapy.
 2. The method of claim 1, whereinthe extracellular concentration of tryptophan is increased in theindividual.
 3. The method of claim 1 wherein the composition isadministered systemically to the individual.
 4. The method of claim 1wherein the composition is in a formulation for controlled or sustainedrelease.
 5. The method of claim 4, wherein the composition formulatedfor controlled or sustained release is suitable for subcutaneousimplantation.
 6. The method of claim 4, wherein the compositionformulated for controlled release is a patch.
 7. The method of claim 1wherein the composition is in a formulation for enteral administration.8. The method of claim 7, wherein the composition formulated for enteraladministration is a capsule or tablet.
 9. The method of claim 8 whereinthe capsule or tablet is enteric coated.
 10. The method of claim 8,wherein the capsule or tablet is formulated for controlled or sustainedrelease.
 11. The method of claim 1 wherein the composition furthercomprises a cytokine.
 12. The method of claim 1 further comprisingadministering a cytokine.
 13. The method of claim 1, wherein thecomposition is in a formulation for topical administration.
 14. Themethod of claim 1, wherein the composition is in a formulation for localadministration.
 15. The method of claim 1, wherein the composition is ina formulation for parenteral administration.